Tetanus toxin-mediated cleavage of cellubrevin inhibits proton secretion in the male reproductive tract: Submitted to Journal of Cell Science. We have previously shown that the vacuolar H+ATPase (PP), located in a sub-population of specialized cells, plays a major role in establishing a luminal acidic environment in the epididymis and proximal part of the vas deferens. Low luminal pH is critical for sperm maturation, the prevention of premature activation of acrosomal enzymes, and for maintaining sperm in a quiescent state during storage in these organs. In the present study, we examined the molecular mechanisms responsible for the regulation of bafilomycin-sensitive proton secretion in the cauda epididymis and vas deferens. We show that in vivo microtubule disruption induces a marked redistribution of H+ATPase from the apical membrane and sub-apical vesicles to numerous intracellular vesicles scattered throughout the cytoplasm, leading to an almost complete loss of H+ATPase polarity. Endocytic vesicles, visualized by Texas red-dextran internalization, contain H+ATPase, indicating active endocytosis of the pump. Cellubrevin, an analog of the v-SNARE synaptobrevin, is highly enriched in PP-rich cells of the epididymis and vas deferens, and tetanus toxin treatment markedly inhibited bafilomycin-sensitive proton secretion by 64.3 1 9.0% in the proximal vas deferens. Western blotting showed effective cleavage of cellubrevin by tetanus toxin in intact vas deferens, demonstrating that the toxin gained access to cellubrevin in this tissue preparation. These results suggest that H+ATPase is actively endocytosed and exocytosed in proton-secreting cells of the epididymis and vas deferens, and that net proton secretion requires the participation of one member of the SNARE fami ly, the v-SNARE cellubrevin. 3) Effect of heavy metals on proton secretion: Exposure to lead, cadmium, manganese and mercury has been shown to reduce male reproductive capacity in a variety of species including human. We are examining whether this reduction of male fertility can be attributed to a defect of the acidification capacity in the epididymis and vas deferens. Using the proton selective probe, we found a marked inhibition of proton secretion in vas deferens accutely exposed in vitro to the heavy metal cadmium (500 5M). This inhibition is of the same amplitude as that observed with either acetazolamide, SITS or bafilomycin, and the latter had no additional effect indicating inhibition of the proton pump by cadmium.